This a 371 of PCT/GB97/02467, filed on Sep. 10, 1997.
The present invention relates to a class of compounds useful in the diagnosis or radiotherapy of infection, inflammation or thrombi, pharmaceutical formulations containing them, their use in diagnosis of disease and methods for their preparation.
Diagnostic imaging of infection or inflammation in clinical practice typically uses either the radiopharmaceutical 67Ga citrate, or radiolabelled white blood cells (leucocytes) since leucocytes are known to accumulate at sites of infection/inflammation. 111In or 99mTc are the radioisotopes normally used to label leucocytes.
Labelled leucocytes have become the method of choice in current clinical practice for the diagnostic imaging of sites of infection/inflammation. With the favourable imaging characteristics of 99mTc, the specificity of labelled leucocytes and good background clearance, this approach lends itself to the diagnosis of gut lesions such as inflammatory bowel disease or appendicitis. Such diseases cannot be diagnosed with non-specific agents due to the high gut background levels. The problem with the ex-vivo labelled leucocyte approach is that the current cell labelling agents used are non-specific. This means that the leucocytes have to be first separated from the excess red blood cells in blood taken from the patient to be imaged. The cell separation is a labour-intensive operation which requires a skilled operator to achieve efficient separation without compromising white cell integrity. There is also the intrinsic hazard associated with manipulation of blood samples. There has therefore been considerable interest in the field in developing an agent which could be used to image sites of infection which does not require this onerous cell separation prior to labelling and administration to the patient.
Human leucocyte elastase (HLE) is a powerful endopeptidase enzyme capable of hydrolysing amide bonds in a variety of proteins and peptides, including the structural proteins elastin, collagen and fibronectin [R L Stein et al, Ann. Rep. Med. Chem., 20, 237 (1985); P D Edwards and P R Bernstein, Med. Chem. Rev., 14, 127 (1994)]. At sites of infection or inflammation HLE is released by the activated leucocytes and causes tissue destruction. It is also known that leucocytes accumulate in thrombi and that elastase is released during blood coagulation. The released elastase has been shown to be important in fibrinogenlysis [E. F. Plow, J. Clin. Invest., 69, 564-572 (1982)].
EP 0595557 A1 (Merck) discloses that the following compounds are useful as HLE inhibitors for the treatment of inflammatory pathologies such as emphysema or rheumatoid arthritis: 
where: R is C1-6 alkyl;
R1 is C1-6 alkyl or C1-6 alkoxy-C1-6 alkyl;
M is H, C1-6 alkyl, hydroxyalkyl, alkoxyalkyl, haloalkyl or C2-6 alkenyl;
Ra and Rb are H, Hal, OH, Ph, COOH or C1-6 alkyl, alkoxy or ester;
R2 and R3 are H, Hal, COOH, Ph, OH, CN, amino, C1-6 alkyl, alkoxy or ester, or amide
R4 is amide or ester with an optional alkyl spacer group.
Radio labelled HLE inhibitors have been little studied. Moser et al. [Am. J. Med., 84 (Suppl.6A), 70 (1988)] report that 131I-labelled xcex11-antitrypsin persists in the human lung for up to one week post injection with little or no uptake in the liver or spleen. The paper does not, however, discuss potential applications of radiolabelled (xcex1-antitrypsin. A tritium-labelled synthetic HLE inhibitor has been used to study the pharmacokinetics of the HLE inhibitor in rats and monkeys [J. B. Doherty et al, Proc. Nat. Acad. Sci. USA, 90, 8727-31 (1993)]. A 13C-labelled synthetic HLE inhibitor has been prepared to study the interaction of the inhibitor with HLE in vitro [B. G. Green et al, Biochem. 34, 14331-14355 (1995)]. Neither publication discloses the use of labelled HLE inhibitors for diagnostic imaging of infection, inflammation or thrombi nor are 3H or 13C suitable labelling moieties for external diagnostic imaging. Rusckowski et al [J. Nucl. Med., abstract P667 (1996)] report that a genetically engineered protein inhibitor of HLE named EPI-hNE-2 (molecular weight 6759) can be radiolabelled with 99mTc via a bifunctional chelate. The radiolabelled protein is reported to show some uptake in a mouse infection model. EPI-hNE-2 is an oligopeptide and could suffer from the same immunogenic problems as Fabxe2x80x2 or larger fragments of antibodies. Furthermore, the size of the molecule limits migration across cell membranes (e.g. those of granulocytes), hence intracellular HLE would not be targeted using this approach.
Blaszczak et al [J. Lab. Comp. Radiopharm., 27, 401-406 (1989)] have described the preparation of 125I-radiolabelled penicillin V (i.e. a bicyclic xcex2-lactam) for use in the in vitro assay of penicillin binding protein. There is no suggestion in the paper of in vivo imaging applications and 125I would not be a preferred radioisotope for external imaging.
It has now been discovered that labelled synthetic HLE inhibitors are useful in the detection of sites of infection or inflammation. Use of a synthetic as opposed to a proteinaceous or polypeptide inhibitor has the significant advantages that the chemical nature of the agent can be fully defined, and potential concerns over immunogenicity are avoided. In addition the position of the label is known unambiguously, and unlike chemotactic peptides or interleukins, the labelled elastase inhibitor is not required to be of very high specific activity because there is an excess of elastase present both within granulocytes and at sites of infection/inflammation. The labelled HLE inhibitors are also useful in the detection of thrombi.
Thus the present invention relates to diagnostic agents for the detection of sites of infection or inflammation or thrombi in the human body. The agents comprise a synthetic human leucocyte elastase (HLE) inhibitor which has a molecular weight of less than 2000 Daltons and is labelled with a detectable moiety suitable for external imaging (e.g. by scintigraphy or MRI), such as a radionuclide or a paramagnetic metal ion. The agent acts by targeting HLE either within leucocytes (in vivo or in vitro), or at sites of HLE release such as sites of infection, inflammation or thrombi. Radiolabelled HLE inhibitors have been shown to selectively label human granulocytes in vitro and to target sites of infection/inflammation in vivo in an animal model of this pathology.
The xe2x80x9cdetectable moietyxe2x80x9d is a substance suitable for external imaging after human administration such as a radionuclide which emits radiation that can penetrate soft tissue; a paramagnetic moiety as a contrast agent for MRI (e.g. certain metal ions such as gadolinium(III), or manganese(II)); a radiopaque moiety such as lopamidol for X-ray contrast imaging (computer assisted tomography ) or an ultrasound contrast agent. Preferably, the detectable moiety is a radionuclide which is either a positron emitter (such as 18F, 11C, 15O, 13N, 68Ga or 64Cu) or a xcex3-emitter such as 123I, 99mTc, 111In, 113 m In or 67Ga. Most preferred radionuclides are xcex3-emitters, especially 123I and 99mTc. 3H and 14C do not have radioactive emissions suitable for external imaging and are therefore outside the scope of the present invention. It is also envisaged that certain radionuclides will confer useful radiotherapeutic properties on the labelled HLE inhibitors. Thus for example 90Y, 89Sr, 186Re, 188Re, 125I or 131I labelled HLE inhibitors could be used in the treatment of rheumatoid arthritis and other bone infections/inflammations. In such applications the therapeutic effect would be due to the local targeted radioactive dose delivered to specific cells, as opposed to any pharmacological effect due to the inhibitor. Whichever detectable moiety is chosen, it is strongly preferred that it is bound to the synthetic HLE inhibitor in such a way that it does not undergo facile metabolism in blood (in vivo or in vitro) with the result that the biodistribution of the detectable moiety no longer reflects that of the HLE inhibitor.
The data presented herein demonstrate that radiolabelled synthetic HLE inhibitors offer a novel and convenient method for targeting sites of infection/inflammation or thrombosis. The mechanism is believed to involve binding to HLE present within circulating granulocytes in the bloodstream (which then concentrate at the site of pathology), or binding to free, extracellular HLE released at sites of pathology. Such a directly-injectable infection/inflammation imaging compound offers significant advantages over existing and proposed radiopharmaceuticals. The specificity for HLE means that the agents should, like ex vivo labelled leucocytes, be capable of imaging lesions associated with leucocyte infiltration such as appendicitis or inflammatory bowel disease. Use of a relatively small synthetic molecule means that the background clearance problems associated with macromolecules are avoided, and substituents can readily be varied in a controlled manner to adjust lipophilicity, plasma protein binding and rate of clearance.
The following classes of synthetic HLE inhibitor with a molecular weight of less than 2000 are suitable for the present invention:
Short chain (3-5 mer) peptides and peptide analogues (e.g. trifluoroacetyl peptides), hydrophobic inhibitors (e.g. elasnin and synthetic analogues).
Inhibitors against the active site histidine (e.g. peptide chloromethyl ketones).
Covalent inhibitors (e.g. peptide aldehydes, peptide ketones, halomethylketones, peptide boronic acids) and acylating agents (sulphonyl fluorides, aminoalkyl phosphono fluoridates, azapeptide nitrophenyl esters, activated carbamates and latent isocyanates, benzoxazin-4-ones, 3-alkoxyl-4-chloroisocoumarins, isatoic anhydrides, acyl saccharins).
Mechanism based inhibitors (e.g. chloropyrones and chloroisocoumarins, 7-amino-4-chloroisocoumarins, ynenol lactones and xcex2-lactams).
Preferred HLE inhibitors for use in the present invention are the mechanism-based inhibitors, especially xcex2-lactams which can be monocyclic (i.e. azetidinones) or have more than one fused ring (such as penicillins, cephalosporins or clavulanic acid analogues) and ynenol lactones. Most preferred are xcex2-lactams, especially monocyclic xcex2-lactams (i.e. azetidinones).
Preferred xcex2-lactams are of formula: 
Where R1 is R8, XR8, (CRR)n(Cxe2x95x90X)R8 or (Cxe2x95x90X)NR82 
X is O or S
n is 0-3.
R8 is H, OH, a substituted or unsubstituted C3-12 carbocyclic or heterocyclic ring which may be saturated or unsaturated, C1-10 alkyl, C3-12 aryl, C4-12 alkylaryl, C2-10 alkenyl, C2-10 alkynyl, C1-10 alkoxy, C1-10 alkoxyalkyl, C1-10 hydroxyalkyl, C1-10 aminoalkyl, C1-10 perfluoroalkyl, C1-10 haloalkyl, C1-10 carboxyalkyl, C1-10 amidoalkyl or C1-10 ketoalkyl.
R2, R3 are the same or different and each H, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C1-4 alkoxyalkyl, C1-4 hydroxyalkyl, C1-4 aminoalkyl, C1-4 perfluoroalkyl, C1-4 haloalkyl, Hal, C1-4 carboxyalkyl, OR, SR, NRR (CH2)nCONRR, NR(CO)R or (CH2)nCO2R.
R4 is a leaving group chosen from Hal, XR8, X(Cxe2x95x90X)R8, OSOR8, OSO2R8 OSO2Hal, SOR8 SO2R8 SO2NR82 NRSO2R, (Cxe2x95x90X)R8, (Cxe2x95x90X)NR82, (Cxe2x95x90X)R8, NO2, CN, POnR82 or XC6H4xe2x88x92n Yn 
Y is the same or different and is R, NO2, Hal, CONR82, SO2NR82 or CO2R.
R5 is R or R4 
R is the same or different and is H, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C1-4 alkoxy, C1-4 alkoxyalkyl, C1-4 hydroxyalkyl, C1-4 aminoalkyl, C1-4 perfluoroalkyl, C1-4 haloalkyl or C1-4 carboxyalkyl.
whereby two or more of the groups R1, R2, R3, R4 and R5 may be combined to form a substituted or unsubstituted carbocyclic or heterocyclic ring which may be saturated or unsaturated,
characterised in that the xcex2-lactam contains or has covalently bonded thereto at least one detectable moiety, and with the proviso that when R4 is XR8, X is S and R1 and R4 are combined to form a cyclic carboxyalkyl group, then the detectable moiety is not 125I.
Preferred azetidinones are of formula: 
Where R1 is R8, XR8, (CRR)n(Cxe2x95x90X)R8 or (Cxe2x95x90X)NR82 
X is O or S
n is 0-3.
R8 is H, OH, a substituted or unsubstituted C3-12 carbocyclic or heterocyclic ring which may be saturated or unsaturated, C1-10 alkyl, C3-12 aryl, C4-12 alkyiaryl, C2-10 alkenyl, C2-10 alkynyl, C1-10 alkoxy, C1-10 alkoxyalkyl, C1-10 hydroxyalkyl, C1-10 aminoalkyl, C1-10 perfluoroalkyl, C1-10 haloalkyl, C1-10 carboxyalkyl, C1-10 amidoalkyl or C1-10 ketoalkyl.
R2, R3 are the same or different and each H, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C1-4 alkoxyalkyl, C1-4 hydroxyalkyl, C1-4aminoalkyl, C1-4 perfluoroalkyl, C1-4 haloalkyl, Hal, C1-4 carboxyalkyl, OR, SR, NRR (CH2)nCONRR, NR(CO)R or (CH2)nCO2R.
R4 is a leaving group chosen from Hal, XR8, X(Cxe2x95x90X)R8, OSOR8, OSO2R8 OSO2Hal, SOR8 SO2R8, SO2NR82 NRSO2R, (Cxe2x95x90X)R8, (Cxe2x95x90X)NR82, (Cxe2x95x90X)R8, NO2, CN, POnR82 or XC6H4xe2x88x92nYn 
Y is the same or different and is R, NO2, Hal, CONR82, SO2NR82 or CO2R.
R5 is R or R4 
R is the same or different and is H, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C1-4 alkoxy, C1-4 alkoxyalkyl, C1-4 hydroxyalkyl, C1-4 aminoalkyl, C1-4 perfluoroalkyl, C1-4 haloalkyl or C1-4 carboxyalkyl.
characterised in that the azetidinone contains or has covalently
bonded thereto at least one detectable moiety.
Attachment of the detectable moiety at the R4 position is least preferred since this group is believed to be lost from the xcex2-lactam as a consequence of covalent binding to the active site of the elastase enzyme. Groups R2 and R3 on the xcex2-lactam are responsible for binding to the S1 site on the enzyme, and literature evidence suggests that these groups need to be relatively small (e.g. the size of an ethyl moiety). Hence it is likely that only a limited range of detectable moieties could be successfully attached at the R2/R3 positions. The detectable moiety is therefore most preferably attached at the R1 position of the xcex2-lactam or azetidinone.
Preferred R1 groups are those of formula: 
where R6 is an optionally substituted alkyl or phenyl group to which the detectable moiety is attached and R7 is H or a C1-6 alkyl group.
When the detectable moiety is a radioactive or paramagnetic metal the metal is always chelated, i.e. a chelate-HLE inhibitor conjugate is used. Metal complexes of the HLE inhibitor alone (i.e. HLE inhibitors without at least one substituent which is designed to coordinate to metal atoms) are not part of the present invention. The term chelate-HLE conjugate covers the situations where the chelating agent is attached as a discrete chemical entity (i.e. as a single substituent on the HLE inhibitor), and when two or more metal donor atoms are attached as substituents at different positions on the HLE molecule. The chelate-HLE inhibitor conjugate is complexed with metal ions (such as technetium, gadolinium or yttrium) giving a metal complex of the chelating agent which is linked to the synthetic HLE inhibitor. The chelating agent is preferably polydentate and/or macrocyclic so that a stable metal complex is formed which can survive challenge by endogenous competing ligands for the metal in vivo such as transferrin or plasma proteins. When intracellular HLE is the target, the metal complex is preferably neutral since this facilitates transport of the labelled inhibitor conjugate across cell membranes such as those of granulocytes. When extracellular HLE released at sites of infection/inflammation or thrombi is the target then membrane permeability is of less importance and a charged metal complex may be desirable to facilitate background clearance. The metal complex should also preferably be of low lipophilicity (since high lipophilicity is often related to non-specific uptake), and exhibit low plasma protein binding (PPB) since plasma-bound label again contributes to undesirable high, non-specific blood background for the imaging agent.
Examples of suitable chelating agents for technetium are diaminedioximes (U.S. Pat. No. 4,615,876) or such ligands incorporating amide donors (WO 94/08949); the tetradentate ligands of WO 94/22816; diaminedithiols, tetraamines or dithiosemicarbazones. Stable technetium complexes are also formed with macrocyclic amine or amide ligands such as cyclam, oxocyclam (which forms a neutral technetium complex) or dioxocyclam. Suitable ligands for indium, yttrium and gadolinium are described in Sandoz WO 91/01144, preferred are macrocyclic aminocarboxylate and aminophosphonic acid ligands. Non-ionic (i.e. neutral) metal complexes of gadolinium are known and examples are described in U.S. Pat. No. 4,885,363.
When the detectable moiety is a radioactive isotope of iodine the radioiodine atom is preferably attached via a direct covalent bond to an aromatic ring such as a benzene ring, or a vinyl group since it is known that iodine atoms bound to saturated aliphatic systems are prone to in vivo metabolism and hence loss of the detectable moiety.
The compounds of the present invention may be prepared as follows:
When the detectable moiety is radioactive iodine, the R1-5 group is chosen to include either a non-radioactive halogen atom (to permit radioiodine exchange), an activated aryl ring (e.g. a phenol group) or an organometallic precursor compound such as a trialkyltin, trialkylsilyl or other such moiety known to those skilled in the art. Examples of suitable R1-5 groups to which radioactive iodine can be attached are given below: 
Both contain substituents which permit facile radioiodine substitution onto the aromatic ring. Alternative substituents containing radioactive iodine can be synthesised by direct iodination via radiohalogen 
When the detectable moiety is a radioactive or paramagnetic metal ion the metal is preferably attached as a metal complex, i.e. a chelating agent is attached to the synthetic HLE inhibitor giving a chelate-HLE inhibitor conjugate. Such chelate-HLE inhibitor conjugates can be prepared using the bifunctional chelate approach. Thus it is well known to prepare chelating agents which have attached thereto a functional group (xe2x80x9cbifunctional chelatesxe2x80x9d). Functional groups which have been attached to chelating agents include: amine, thiocyanate, maleimide and active ester such as N-hydroxysuccinimide. Such bifunctional chelates can be reacted with suitable functional groups on the HLE inhibitor to form the desired conjugate. Examples of chelate-amine conjugates for diaminedioxime ligands are given in WO 95119187. In the particular case of xcex2-lactams, a chelating agent can be attached at the R1 position as follows. First a chelate-amine conjugate is converted to a chelate-isocyanate conjugate using phosgene, trichloromethylchloroformate or similar. The chelating agent may optionally be protected with protecting groups known to those skilled in the art. The resulting chelate-NCO (isocyanate) conjugate can then be reacted with the amine NH of an azetidinone ring secondary amine giving a chelating agent attached at R1 via a urea linkage. Similarly, a chelate-amine conjugate can be converted to a chelating agent with a pendant isothiocyanate group (as e.g. described in U.S. Pat. No. 5006643 or WO 91/01144) and then reacted with an azetidinone to give a chelating agent-azetidinone conjugate linked via a thiourea bond. Alternatively, reaction of a chelate-active ester conjugate with the amine NH of an azetidinone ring would give a chelate-azetidinone conjugate linked via an amide bond. A further approach to an amide-linked conjugate would be to couple the amine group of an chelate-amine conjugate to the pendant carboxyl group of a carboxyl-functionalised azetidinone. Persons skilled in the art will recognise that many alternative syntheses of chelate-HLE inhibitor conjugates are possible based on this disclosure.
The present invention also relates to kits for the preparation of synthetic HLE inhibitors labelled with a detectable moiety. The kits are designed to give sterile products suitable for human administration, e.g. via injection into the bloodstream. Possible embodiments are discussed below. When the detectable moiety is 99mTc, the kit would comprise a vial containing the chelate-HLE inhibitor conjugate together with a pharmaceutically acceptable reducing agent such as sodium dithionite, sodium bisulphite, ascorbic acid, formamidine sulphonic acid, stannous ion, Fe(II) or Cu(I), preferably a stannous salt such as stannous chloride or stannous tartrate. Alternatively, the chelating agent-HLE inhibitor conjugate could be present as a metal complex which, upon addition of the radiometal, undergoes transmetallation (i.e. ligand exchange) giving the desired product. The kit is preferably lyophilised and is designed to be reconstituted with sterile 99mTc-pertechnetate (TcO4xe2x88x92) from a 99mTc radioisotope generator to give a solution suitable for human administration without further manipulation.
The agents of the present invention may also be provided in a unit dose form ready for human injection and could for example be supplied in a pre-filled sterile syringe. When the detectable moiety is a radioactive isotope such as 99mTc, the syringe containing the unit dose would also be supplied within a syringe shield (to protect the operator from potential radioactive dose).
The above kits or pre-filled syringes may optionally contain further ingredients such as buffers; pharmaceutically acceptable solubilisers (e.g. cyclodextrins or surfactants such as Pluronic, Tween or phospholipids); pharmaceutically acceptable stabilisers/antioxidants (such as ascorbic acid, gentisic acid or para-aminobenzoic acid) or bulking agents for lyophilisation (such as sodium chloride or mannitol).
The structures of particular compounds 1-52 are set out below. Preparation of these compounds is described in Examples 1 to 12. NMR data for the compounds is given in Tables 1 to 19. Biological properties of compounds 4, 4a, 4b, 16, 17, 24, 28, 38, 42, 48, 49, 51 and 52 are shown in Examples 13 to 17 and in Tables 20 to 25.
In one aspect of the present invention, the desired agent is required to label human granulocytes selectively in whole blood (either in vitro or in vivo). In normal human blood erythrocytes outnumber granulocytes by a factor of at least 1000:1 but erythrocytes do not accumulate at sites of infection/inflammation hence the labelled compound must exhibit high selectivity for granulocytes. Studies on the in vitro human blood cell uptake of 123I-labelled and 99mTc-labelled xcex2-lactam HLE inhibitors have demonstrated selective uptake in human granulocytes (see Example 14 and Table 23). Table 23 shows that compounds 4, 4a, 4b, 16, 17, 24, 28, 38, 42, 48, 49, 51 and 52 all show some degree of selectivity for granulocytes over a mixture of monocytes/lymphocytes when approximately equivalent numbers of monocytes/lymphocytes and granulocytes are present. Since human granulocytes contain significant levels of HLE whereas erythrocytes (red blood cells), monocytes and lymphocytes (subsets of the leucocyte cell population) contain essentially no HLE, this selectivity is a strong indication that affinity for HLE is involved. Furthermore, Table 23 shows that there is a correlation between the in vitro potency (measured for the non-radioactive iodine-labelled compounds and for the unlabelled chelate conjugates, compounds 38, 42, 48, 49, 51 and 52) and selectivity for granulocytes. The implication is that retention within the granulocyte is due to binding to intracellular elastase. Preferred synthetic HLE inhibitors of the present invention are therefore those with an in vitro potency (kinact/Ki) of greater than 10,000 M/sec.
Whole blood also contains plasma proteins which are capable of binding to a wide range of substances and hence may compete effectively for the compound once it is introduced into the blood. Hence the preferred agent should also exhibit low plasma protein binding (PPB). Preferred compounds have a PPB of less than 95%. Most preferred have a PPB of less than 60%. Since lipophilic compounds are particularly susceptible to non-specific plasma protein binding, preferred compounds have an octanol/water partition coefficient (LogP) of xe2x89xa62. When intracellular HLE is the target and consequently the agent must be capable of crossing cell membranes, compounds with a minimum octanol/water partition coefficient (P) of 0.3 are preferred. Table 23 shows that, in human whole blood, compounds 4, 16 and 24 exhibit equivalent uptake in granulocytes and erythrocytes. This implies a selectivity factor for granulocytes over erythrocytes of at least 1000:1 and shows that the compounds of the present invention are capable of successfully labelling granulocytes in human whole blood despite the competition from excess erythrocytes and plasma proteins. It is believed that the superior granulocyte selectivity of compound 4 may be due to a combination of HLE potency and reduced non-specific binding to red blood cells or plasma proteins. It is postulated that the amine substituent facilitates selective retention in granulocytes due to diffusion of the uncharged inhibitor into the cell and its subsequent trapping via protonation of the basic piperazine amine within the more acidic milieu of the azurophil granule of the granulocyte. The protonated inhibitor cannot readily diffuse back across the granulocyte membrane. Such more acidic environments are not present within erythrocytes, monocytes or lymphocytes.
By virtue of the single substituent at the 4 position of the azetidinone ring, compound 4 has a chiral centre. The enantiomers were resolved (Example 1, step F) and one (4a) was found to exhibit markedly superior in vitro potency and granulocyte selectivity (Table 23) compared to the other (4b). Thus efficacy is highly sensitive to stereochemical effects, i.e. chirality. Therefore chiral compounds are also encompassed by the present invention.
Further improvements in potency have been achieved by the introduction of alkyl substituents at the homochiral benzylic R1 position, demonstrated by compounds 24 and 28, since these are known to give more potent HLE inhibitors (EP 0595557 A1) e.g.: 
The radiolabelled xcex2-lactam HLE inhibitors of the present invention have also been studied in vivo in a rat model of inflammation/infection. The results on such studies are given in Table 24. The known inflammation/infection agent 99mTc-HMPAO ex-vivo labelled human leucocytes was shown to locate at the site of infection in the model used. 99mTc-red blood cells was used as the negative control agent comparison. It can be seen that 123I-labelled compounds 4/4a show significantly better uptake in the infected region than the 99mTc-rbc control, and have characteristics which more closely resemble the proven infection agent 99mTc-wbc. When 123I-compound 4 is used to label human leucocytes ex-vivo the infected/normal ratio is higher than that obtained by direct injection. It is believed that the target to background ratios obtained in the rat model of infection/inflammation almost certainly underestimate the human situation because the potency of similar xcex2-lactam inhibitors for rat elastase is known to be up to 2 orders of magnitude less than that for human leucocyte elastase.
Experiments with human plasma clots in vitro have been performed as described in Example 17. In clots enriched with granulocytes to a final concentration of 106/ml there was an approximate 7-9 fold increase in the uptake of a potent elastase inhibitor compared to those clots formed without added cells. This observation indicates the potential for radiolabelled xcex2-lactam HLE inhibitors to be specifically taken up in thrombi and other lesions where granulocyte accumulation is active.
Table 25 compares the thyroid uptake of the radioiodine compounds of the present invention with that of free 123I-iodide ion. The lack of thyroid uptake is evidence that the compounds do not undergo in vivo metabolic de-iodination. There was no evidence either for the release of pertechnetate in animals dosed with 99mTc-labelled HLE inhibitors. This implies that the technetium is not released from the chelate conjugate in vivo.